Flycatcher Song in Allopatry and Sympatry – Convergence, Divergence and Reinforcement

doi:10.1111/j.1420-9101.2003.00682.x

Flycatcher song in allopatry and sympatry – convergence, divergence and reinforcement

J. HAAVIE,* T. BORGE,* S. BURES,à L. Z. GARAMSZEGI,§ H. M. LAMPE, J. MORENO,– A. QVARNSTRO¨ M,** J. TO¨ RO¨ K & G.-P. SÆTRE* *Department of Evolutionary Biology, Evolutionary Biology Centre, Uppsala University, Uppsala, Sweden Department of Biology, University of Oslo, Oslo, Norway àLaboratory of Ornithology, Palacky University, Olomouc, Czech Republic §Department of Biology, Universitaire Instelling Antwerpen, Wilrijk, Belgium –Departamento de Ecologı´a Evolutiva, Museo Nacional de Ciencias Naturales, Madrid, Spain **Department of Animal Ecology, Evolutionary Biology Centre, Uppsala University, Uppsala, Sweden Behavioural Ecology Group, Department of Systematic Zoology and Ecology, Eo¨tvo¨s University, Budapest, Hungary

Keywords: Abstract cultural inheritance; The theory of reinforcement predicts that natural selection against the Ficedula; production of unfit hybrids favours traits that increase assortative mating. hybridization; Whether culturally inherited traits, such as bird song, can increase assortative reinforcement; mating by reinforcement is largely unknown. We compared songs of pied song; (Ficedula hypoleuca) and collared flycatchers (F. albicollis) from two hybrid species recognition. zones of different ages with songs from allopatric populations. Previously, a character divergence in male plumage traits has been shown to reinforce premating isolation in sympatric flycatchers. In contrast, we find that the song of the pied flycatcher has converged towards that of the collared flycatcher (mixed singing). However, a corresponding divergence in the collared flycatcher shows that the species differences in song characters are maintained in sympatry. Genetic analyses suggest that mixed song is not caused by introgression from the collared flycatcher, but rather due to heterospecific copying. Circumstantial evidence suggests that mixed song may increase the rate of maladaptive hybridization. In the oldest hybrid zone where reinforcement on plumage traits is most pronounced, the frequency of mixed singing and hybridization is also lowest. Thus, we suggest that reinforcement has reduced the frequency of mixed singing in the pied flycatcher and caused a divergence in the song of the collared flycatcher. Whether a culturally inherited trait promotes or opposes speciation in sympatry may depend on its plasticity. The degree of plasticity may be genetically determined and accordingly under selection by reinforcement.

Introduction population will mix (introgression) and over time the populations will blend. However, if the hybrids have low When two populations come into secondary contact they ﬁtness, natural selection will favour traits that increase may have diverged to such an extent that they do not assortative mating, thereby reducing the frequency of interbreed (prezygotic barrier) or their hybrid offspring maladaptive hybridization (Dobzhansky, 1937; Dobzhan- do not survive or reproduce (post-zygotic barrier). If pre- sky, 1940). This process, known as reinforcement, will and post-zygotic barriers are incomplete, genes from each cause signals used in mate recognition to diverge, thereby reinforcing prezygotic barriers (Sætre et al., 1997b) Correspondence: Jon Haavie, Department of Evolutionary Biology, eventually completing speciation in sympatry. Thus, Evolutionary Biology Centre, Uppsala University, Nordbyva¨gen 18D, SE-752 36 Uppsala, Sweden. depending on the ﬁtness of the hybrid, prezygotic Tel.: +46 18 471 2640; fax: +46 18 471 6310; barriers will either increase or decrease (Parsons et al., e-mail: [email protected] 1993; Sætre et al., 1997b; de Kort et al., 2002).

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Prezygotic barriers are generally assumed to be genetically determined (Dobzhansky, 1937). However, premating barriers may develop for nongenetical reasons (Grant & Grant, 1997a). Song in oscine birds is usually learned through an imprinting-like process (Kroodsma, 1982; Catchpole & Slater, 1995). This property may cause song to change rapidly over time and vary geographically (for references see Catchpole & Slater, 1995). It has been suggested that such culturally inherited traits can increase assortative mating and possibly be the starting point for speciation in allopatry (Marler & Tamura, 1962; Baker & Mewaldt, 1978). However, when two closely related species come into secondary contact males may come to copy songs of the wrong model (Helb et al., 1985). Consequently, heterospecific copying may lead to hybridization (Alatalo et al., 1990; Eriksson, 1991; Grant & Grant, 1997c). Whether reinforcement acts on culturally inherited traits to increase assortative mating is largely unknown (Grant & Grant, 1997b). Thus, it is unclear whether culturally inherited traits promote or oppose speciation and the role of song in speciation remains controversial (Raikow, 1986; Baptista & Trail, Fig. 1 The European distribution of the pied flycatcher (grey area) 1992; Salomon & Hemim, 1992; Grant & Grant, 1997a; and collared flycatcher (hatched area). Song recordings are of Irwin & Price, 1999; Irwin et al., 2001; Slabbekoorn & allopatric pied flycatchers from Spain near Madrid (SPA) and Norway Smith, 2002). By comparing traits of populations where near Oslo (NOR), collared flycatchers from Italy in Abruzzo National their distribution overlaps (sympatry) with populations Park (ITA) and Hungary near Budapest (HUN). Sympatric popula- where they live separately (allopatry) we can examine tions of pied and collared flycatchers were from the Baltic island of the effects of coexistence on traits used in species O¨ land, Sweden (SWE) (referred to as the Swedish populations) and recognition. In this way it may be possible to study the Czech Republic in the Jesenı´k mountains (CZE). whether culturally inherited traits promote or oppose speciation. The pied flycatcher (Ficedula hypoleuca, Pall.) and the isolation in a Central European hybrid zone (Sætre et al., collared flycatcher (F. albicollis, Temm.) have overlapping 1997b). The hybrid zone in Central Europe is assumed to breeding distributions in Central and Eastern Europe and be of secondary contact after the Pleistocene glaciations on the Baltic islands of Gotland and O¨ land off the (von Haartman, 1949; Sætre et al., 2001) whereas the Swedish east coast (hereby referred to as the Swedish Swedish hybrid zone is apparently of more recent origin, population) (Fig. 1). They are closely related species with possibly only about 150 years old (Alatalo et al., 1990). genetic distance estimated at approximately 3% at Interestingly, the species differences in male plumage mitochondrial DNA (Sætre et al., 2001). Also, they are characters are more pronounced and the frequency of ecologically very similar as they compete over nest sites hybridization is lower in the Central European than in and territories in sympatry and affect each other’s the Swedish hybrid zone (Alatalo et al., 1994; Sætre et al., population dynamics (Sætre et al., 1999b). The frequency 1999a, 2003). As plumage traits are used in species of hybridization has been estimated at 2–7% in different recognition, this suggests that the prezygotic barriers sympatric populations (Alatalo et al., 1982; Sætre et al., have evolved further in the Central European than in the 1999a; Veen et al., 2001), and hybrids have greatly Swedish hybrid zone (Sætre et al., 1999a). reduced fitness, consistent with Haldane’s rule (Alatalo The songs of the pied and collared flycatcher are et al., 1982; Sætre et al., 1997b, 1999a, 2003). The pied complex, repertoire sizes typically ranging from a few up and the collared flycatcher have a large overlapping to 100 unique song syllables (Lundberg & Alatalo, 1992), breeding distribution (Fig. 1). However, hybridization but easily distinguishable in the field mainly through occurs in a rather narrow zone corresponding to topog- difference in frequency and tempo (Gelter, 1987). In raphy and habitat (Alerstam et al., 1978; Sætre et al., flycatchers, song has been shown to function in territory 1999a). Females of the two species are similar to the defence (Eriksson, 1991) and mate attraction (Eriksson, extent that males of both species are apparently unable to 1986). Females have been shown to base their mate distinguish between them (Sætre et al., 1997a). However, choice on repertoire size (Eriksson, 1991; Lampe & Sætre, female mate choice is not random (Alatalo et al., 1982; 1995) and song activity and complexity has been found Sætre et al., 1997b) and character displacement on male to correlate with the health status and condition of the plumage colour has been found to reinforce premating male (Garamszegi et al., 2003; Lampe & Espmark, 2003).

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Both character convergence and divergence have been allopatric collared flycatchers were from Italy (41°49¢N, suggested to occur in the songs of flycatchers. In 13°47¢E) and Hungary (47°43¢N, 19°01¢E) (we classified sympatric populations some pied flycatchers sing a song the Hungarian population as allopatric, but note that the resembling that of the collared flycatcher (Tischler, 1942; pied flycatcher is an occasional, rare breeding bird in this Lo¨ hrl, 1955; Gelter, 1987; Eriksson, 1991), a phenom- area). Sympatric populations were from Sweden enon known as mixed song (Eriksson, 1991). This has (57°10¢N, 16°58¢E) and the Czech Republic (49°49¢N, been suggested to be an example of character conver- 17°10¢E) (see Fig. 1 and Table 1 for further details). gence in the pied flycatcher (Eriksson, 1991). However, Species were identified by a combination of DNA mark- Eriksson was not able to study character changes in the ers, plumage characters and species-specific alarm calls song of the collared flycatcher as no allopatric collared (data not shown). Hybrids were excluded from the song flycatcher population was used in the comparisons. By analyses. contrast Wallin (1985) argued for a divergent character The songs were recorded with Sony DAT recorders displacement among sympatric collared flycatchers. (TCD-D7 and TCD-D10), a minidisk recorder (Kenwood However, the comparison was flawed. It was based on DMC-J7R) or a Sony Tape recorder (TC-D5 PRO II). For a very limited sample size; allopatric collared flycatchers recording we used a mono (Classic), a stereo (Science represented with only one individual which has later and PRO II) and a shotgun microphone mounted on a been shown to belong to a distinct species – the 55 cm parabolic dish (all products by Telinga, Tobo, semicollared flycatcher (F. semitorquata) (Sætre et al., Sweden). The songs were analysed in Avisoft-SASLab Pro 1997b, 2001). To clarify the issues of possible character (version 3.93, R. Specht) (Avisoft Bioacoustics, Berlin, convergence and divergence in the song in the Ficedula Germany) using automatic parameter measurement with flycatchers, we conducted a large-scale comparison of standard settings. To increase sensitivity, certain param- song characteristics using four allopatric and two symp- eters were manually adjusted (automatic threshold rel- atric populations. Also, we compared two hybrid zones ative to maximum set between )25 and )20 dB). with different population structures and assumed age of Syllables were defined as continuous elements or groups contact (Sætre et al., 2003). Based on previous evidence of elements separated by noticeable time intervals on the on plumage colour evolution (see above) we predicted sonograms (hold time set between 0.01 and 0.02 s). We that adaptive changes in song are likely to be more separated songs by a minimum interval of 1 s. For each pronounced in the Central European compared with the syllable in the song, start and end time, as well as island hybrid zones. Finally, we applied genetic analysis maximum and minimum frequency were measured. to assess whether gene flow across the partial species From these basic measurements new variables were barriers could affect the song characters in the sympatric calculated. These were: song duration and duration of populations. each syllable (to nearest 0.001 s), number of syllables per song, lowest frequency, highest frequency and frequency Materials and methods range (to nearest 1 Hz) and song tempo (calculated as the average number of syllables per second). Incomplete Song recordings were obtained during April–June 1999– songs (consisting of less than three syllables) and entice- 2002. Allopatric pied flycatchers were from Spain ment calls (songs with more than 20 syllables) were (41°04¢N, 3°27¢W) and Norway (59°55¢N, 10°46¢E); excluded from the analyses. A male flycatcher switches

Table 1 Mean values and standard deviations for the seven song variables of the pied and collared ﬂycatcher in sympatric and allopatric populations. No. of songs refers to the average number of songs analysed per individual.

Species Collared ﬂycatcher Pied ﬂycatcher

Population type Allopatry Sympatry Allopatry Sympatry

Italy Hungary Sweden Czech R Norway Spain Sweden Czech R

Populations variable x SD x SD x SD x SD x SD x SD x SD x SD

Song duration 2.75 0.65 3.18 0.46 3.00 0.63 2.96 0.73 1.86 0.29 1.88 0.38 1.98 0.32 2.14 0.34 No. of syllables 9.39 2.47 11.19 1.79 9.39 2.12 9.82 2.74 9.88 1.60 6.70 1.23 8.04 1.78 10.84 2.07 Low frequency 4101 260 4418 149 4962 191 4830 177 3525 157 3282 265 4212 392 3800 260 High frequency 5806 284 5800 148 6196 259 6105 202 5417 179 5488 170 5742 390 5383 305 Frequency difference 1705 348 1382 177 1235 175 1275 91 1892 152 2206 158 1530 261 1584 275 Syllable duration 0.17 0.03 0.15 0.01 0.20 0.02 0.17 0.01 0.11 0.01 0.18 0.01 0.16 0.02 0.12 0.01 Tempo 3.45 0.27 3.55 0.22 3.15 0.33 3.32 0.19 5.36 0.33 3.65 0.24 4.09 0.46 5.08 0.54 No. of songs 28.2 12.3 32.0 15.6 26.2 6.1 21.0 9.4 40.7 18.7 19.5 8.3 32.5 13.9 24.8 8.4 n 26 14 17 10 30 6 8 20

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F. albicllis – Italy

F. albicllis – Hungary

F. albicllis – Czech Rep.

F. albicllis – Sweden

F. hypoleuca – Sweden – mixed song

F. hypoleuca – Sweden – pure song

F. hypoleuca – Czech Rep.

F. hypoleuca – Norway 10 kHz 5 1 s F. hypoleuca – Spain Fig. 2 Sonograms of pied and collared flycatchers from the study populations. to enticement calls when a female visits his nest hole. earlier studies that found them useful in characterizing This song type is therefore probably not directly compar- the species (Wallin, 1985; Gelter, 1987). To compare able with normal territorial song. Mean values for each overall similarity between the species and the popula- individual were calculated for all measurements. The tions, the data were analysed in a principal component choice of measurements in this study was based on analysis and represented in scatterplots. One variable, the frequency range, was excluded from the principal component analysis because of low extraction commonality. Furthermore, the data were analysed in a discriminant function analysis. Convergence or divergence in the song characters was tested by ANOVAs. To compare population differences in song content, we made sonograms (see examples from the different populations in Fig. 2), and classified each syllable into one of five categories: note, vibrato, figure, figure/vibrato and figure/note (see Fig. 3 for details). The classifications were made blindly, that is, without knowing the species or population origin of the Fig. 3 The classification of syllables based on five categories. These categories were (a) note, (b) vibrato, (c) simple figure, (d) figure birds. The relative frequencies of syllables in each with a vibrato and (e) figure with a note. The two last categories category were calculated to allow population compari- defined syllables with elements that were produced either in sons. sequence or simultaneously, independent of the order. The fre- Genetic data on birds from the sympatric population of quencies of the classified syllables are presented in Fig. 6. O¨ land include genotypes at 21 genetic markers that have

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been used to estimate the proportion of introgressed Table 2 Correlation matrix of the variables and the first two alleles in the genomes of the individual birds (Sætre et al., components in the principal component analysis (PC1 and PC2) and 2003). For the present study, we compared these genetic the standardized canonical discriminant function coefficients (di). results with the data on song, to investigate whether Variable PC1 PC2 di introgressive hybridization would affect the song of sympatric birds. Song duration 0.55 0.80 )0.44 No. of syllables )0.23 0.97 0.09 Low frequency 0.86 0.20 )0.68 Results High frequency 0.85 0.11 )0.07 Syllable duration 0.91 )0.15 0.42 The pied and the collared flycatcher were found to differ Tempo )0.93 0.03 1.01 in several song characters in both sympatry and allopatry (Table 1). Qualitatively, the song of the collared flycatcher can be described as having longer songs, a lower tempo but a higher frequency (both minimum and (Fig. 4). A high PC1 score is associated with longer maximum frequencies) than that of the pied flycatchers. syllables and song duration, lower tempo and higher To simplify the comparisons the data were analysed in a minimum and maximum frequencies (Table 2). Further, principal component analysis. a high PC2 score is associated with more syllables in the The two first principal components explained about song and also longer songs. 86% of the variation in the data and are presented in the Collared flycatchers exhibited higher PC1 scores in scatterplots of allopatric and sympatric populations sympatry than in allopatry (Figs 4 and 5a). Whereas the song variation largely coincides with allopatric vs. sympatric distributions in the collared flycatcher, it appears to (a) 3 be more complex in the pied flycatcher. In the pied flycatcher the allopatric Norwegian and the sympatric 2 Czech populations both exhibited low PC1 scores. Also, the allopatric Spanish and the sympatric Swedish popu-

1 lations exhibited higher PC1 scores and were both quite similar to the allopatric collared flycatchers. For PC2 only Collared Italy minor and nonsignificant intraspecific variation was 0 Collared Hungary observed at the population level. Pied Norway Some sympatric pied flycatchers got PC1 scores more –1 Pied Spain similar to those of collared flycatchers than to allopatric pied flycatchers (Fig. 4). We performed a discriminant –2 analysis to classify sympatric pied flycatchers as singing pied or collared flycatcher song. The relative contribution –3 of the variables to the overall discrimination is presented –2 –1012 in Table 2. Allopatric pied and collared flycatcher songs PC1 were used as templates for calculating the discriminant (b) function. In the discriminant analysis, eight of 28 (29%) 3 sympatric pied flycatchers were classified as singing collared flycatcher songs. These so-called mixed singers 2 (Eriksson, 1991) were much more frequent in the Swedish (six of eight) than in the Czech samples (two 2 1 of 20) (v ¼ 8.859, n ¼ 28, P < 0.05). All sympatric Collared Sweden collared flycatchers were classified to the right species. Collared Czech R. 0 In sympatry, individuals classified as mixed singing pied PC2 PC2 Pied Sweden flycatchers differed from pure singing pied flycatchers in Pied Czech R. five of the seven song characters (Table 3). Four of these –1 five characters also differed between the allopatric and sympatric collared flycatchers. –2 About 88% of the song recordings of individual birds were of such quality that they allowed classification of –3 the syllables in the song. Pied and collared flycatchers –2 –1012 differed in the relative frequency of the five different PC1 classes of syllables (Fig. 6). Syllables classified as figure/ Fig. 4 Principal component scores of individuals from (a) allopatric notes were found almost exclusively in the collared and (b) sympatric pied and collared flycatcher populations. flycatcher. Also, the vibratos and notes were much more

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Table 3 Mean values and one-way ANOVAs of song variables comparing sympatric pied ﬂycatcher scored as singing pure and mixed song in the discriminant analysis, and collared ﬂycatcher from allopatry and sympatry.

Pied ﬂycatcher Collared ﬂycatcher

Pure Mixed Allopatry Sympatry

Variable xxd.f. FP-value xxd.f. FP-value

Song duration 2.12 2.03 1,26 0.41 0.527 2.90 2.98 1,65 0.28 0.601 No. of syllables 10.86 7.99 1,26 12.13 0.002 10.02 9.55 1,65 0.62 0.433 Low frequency 3749 4339 1,26 38.70 <0.001 4212 4913 1,65 132.97 <0.001 High frequency 5364 5789 1,26 10.52 0.003 5804 6162 1,65 35.51 <0.001 Frequency difference 1616 1450 1,26 2.31 0.141 1592 1250 1,65 24.67 <0.001 Syllable duration 0.12 0.16 1,26 31.13 <0.001 0.16 0.19 1,65 21.60 <0.001 Tempo 5.12 3.96 1,26 40.76 <0.001 3.48 3.21 1,65 15.65 <0.001

(a) 2 common in the collared than in the pied flycatcher song. These three classes of syllables comprised 53–82% in the collared flycatcher populations and between 3–48% in pied flycatchers (Fig. 5b). The results of the sonogram 1 analysis were qualitatively similar to that of the principal component analysis of song characters (Fig. 5a and b).

PC1 However, the Spanish pied flycatchers did not differ from 0 Swedish pied flycatchers in the first principal component (Fig. 5a), but differed in the frequency of syllable types (Fig. 5b) being more similar to the Norwegian and Czech populations. Pied flycatchers classified as mixed singers –1 in the discriminant analysis had 60% (the Swedish population) and 44% (the Czech population) of their syllables classified as typical of the collared flycatcher

–2 (Fig. 6). The relative frequency of these three syllable Italy Hungary Czech Sweden Sweden Czech Norway Spain types were found to be significantly higher in sympatric Allopatry Sympatry Sympatry Allopatry than in allopatric collared flycatchers (t ¼ 6.24, d.f. ¼ 56, Collared Pied P < 0.001), whereas no difference was found between allopatric and pure singing sympatric pied (b) 1 flycatchers (t ¼ 1.46, d.f. ¼ 48, n.s.). Mixed singing pied flycatchers did not differ from allopatric collared flycatchers (t ¼ )0.14, d.f. ¼ 40, n.s.), but differed from sympatric collared flycatchers (t ¼ )5.44, d.f. ¼ 30, 0,75 P < 0.001) and pure singing pied flycatchers (t ¼ 9.44, d.f. ¼ 55, P < 0.001). Both genetic and song data was available for five of the 0,5 pied flycatchers and nine of the collared flycatchers from CF like the sympatric Swedish population. Four of the pied flycatchers were classified as mixed singers in the discriminant analysis. However, none of the pied fly- 0,25 catchers possessed any alleles introgressed from the collared flycatcher. We found no difference in song characteristics of five pure collared flycatchers compared 0 with four collared flycatchers carrying at least one allele Italy Hungary Czech Sweden Sweden Czech Norway Spain introgressed from the pied flycatcher (PC1: t ¼ 0.07, Allopatry Sympatry Sympatry Allopatry d.f. ¼ 7, n.s.). Collared Pied

Fig. 5 (a) Mean and 95% confidence intervals of the first principal Discussion component (PC1) and (b) the relative frequency of the three syllable types most typical for collared flycatchers (CF like) of pied and The theory of reinforcement suggests that natural selec- collared flycatcher populations. tion against the production of unfit hybrids should

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Fig. 6 The frequencies of syllable types of pied and collared flycatchers in allopatry and sympatry. Following the discriminant function analysis (see main text for further details), sympatric pied flycatchers have been grouped into mixed and pure singers. Syl- lable types that are typical in the collared flycatcher song have been marked with an asterisk (*). The frequencies of these syllables are presented in Fig. 5b). favour traits that increase assortative mating. Given that characters. Natural selection, such as adaptation to a hybridization is maladaptive and that song is a trait used different acoustical environment, sexual selection or in species recognition, the species differences in song random changes may cause population differences in characters should be larger in sympatry than in allopatry. song (West-Eberhard, 1983; Andersson, 1994; Butlin, The song analysis shows that there are quite clear species 1995; Jones, 1997; Price, 1999). Hence, it does not differences between pied and the collared flycatcher in necessarily follow that differences in signals between allopatry. In sympatry some pied flycatchers resemble allopatric and sympatric populations are because of collared flycatchers in their song, known as mixed song species interactions. In sympatric populations pied and (Eriksson, 1991). However, the species differences are collared flycatcher compete over resources (Sætre et al., maintained in sympatry because of a corresponding 1999b) and hybridize (Alatalo et al., 1982; Sætre et al., divergence in the collared flycatcher. Accordingly, the 1999a; Veen et al., 2001). Hybrids have been found to pattern of song variation can better be described as a have greatly reduced fitness (Alatalo et al., 1982; Sætre character shift than a character displacement. et al., 1997b, 1999a, 2003). A character displacement on The pied flycatcher shows a complex pattern of male plumage traits has been found to reinforce species geographic variation in song that warrants further barriers (Sætre et al., 1997b). The character displacement explanation. First, the frequency of mixed song is higher on plumage traits is a manifestation of the strength of in the Swedish than in the Czech sample of pied species interaction suggesting that it could also affect flycatchers. Secondly, in the principal component analy- other traits such as the song of the flycatchers. sis the two allopatric pied flycatcher populations Our DNA analyses suggest that social interspecific appeared to differ from each other. The Spanish popu- interaction rather than hybridization (per se) has affected lation seems to be intermediate between the Norwegian the song of the flycatchers. Mixed singing has been population and the collared flycatcher (Figs 4a and 5a). described in a number of bird species (Helb et al., 1985). In the classification of syllables, however, no differences However, only the recent application of DNA markers between the allopatric pied flycatcher populations were has made it possible to investigate whether the mixed evident (Fig. 5b). This implies that Spanish pied flycatch- singers are hybrids, backcrosses or individuals with pure ers are similar to the other pied flycatchers in song parental genotype that learn heterospecific song (Bensch content, e.g. the relative frequency of the syllables in the et al., 2002). The comparison of genetics and song shows songs, but resemble collared flycatchers in quantitative that none of the mixed singing pied flycatchers examined traits such as in syllable duration and tempo. Thus, the possessed any alleles introgressed from the collared resemblance of the Spanish pied flycatcher to the collared flycatcher, thus they are not backcrosses. In fact, intro- flycatcher in the principal component analysis is not gression has been found to occur nearly exclusively from because of mixed singing. pied to collared flycatchers and not vice versa (Sætre Allopatric and sympatric populations may differ for et al., 2003). This implies that heterospecific learning is many reasons, but it is likely that species interactions the dominant proximate factor explaining the occurrence have affected pied and collared flycatcher in multiple of mixed song in the pied flycatcher. In accordance with

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this, individual pied flycatchers have been observed zones. Also, mixed song in the pied flycatchers is more singing pure song early in the season but changing to frequent in the Swedish than in the Czech population. mixed song later in the season (Lo¨ hrl, 1955). Thus, social Consequently, the species differences in song are larger interaction with the collared flycatcher is presumably the in the Czech than in the Swedish hybrid zone; a pattern cause of mixed singing (Eriksson, 1991). resembling that found in male plumage variation. Mixed If song is affected by interspecific interactions it could song has been suggested to increase the probability of be expected to differ in the two hybrid zones. The two hybridization (Alatalo et al., 1990; Eriksson, 1991). hybrid zones differ in colonization history, population Accordingly, a plausible explanation for the difference structure and age and this gives different predictions on between the hybrid zones in frequency of mixed song is the extent of adaptations to the coexistence in the pied that mixed singing is maladaptive and that the Central and collared flycatchers. The hybrid zones on the Swedish European population has developed better mechanisms Baltic islands are supposedly much younger than the one to reduce the likelihood of heterospecific song learning. in Central Europe. Thus, any adaptations to coexistence, Also, mixed singing in the pied flycatcher may have such as reinforcement of prezygotic barriers, would have caused a selection pressure for divergence of song had less time to evolve on the Swedish islands than in characters in the collared flycatcher. Interestingly, our Central Europe (Lundberg & Alatalo, 1992). Moreover, results show that the collared flycatchers in sympatry the two classes of hybrid zones also differ in dynamics and have diverged in the same characters that mixed singing structure (Sætre et al., 1999a). The island populations pied flycatchers copy. If a reduction in the frequency of consist of collared flycatchers evidently isolated from any mixed singing and a divergence in the song of the continental population, whereas there would be exten- collared flycatcher increases assortative mating and thus sive gene flow of allopatric pied flycatchers from the decreases the frequency of hybridization, this would be a surrounding mainland. The pattern of gene flow is form of reinforcement. presumably more symmetrical in the continental hybrid An alternative explanation could be that the probability zone (Sætre et al., 1999a; S. Bures, M. Kral, G.-P. Sætre, of developing heterospecific song depends on the relative unpublished data). Thus, any local adaptation in the pied magnitude of exposure to con- and heterospecific song flycatcher population on the Swedish islands is likely to (Becker, 1982). When mixed singing occurs, it is usually be more strongly counteracted by gene flow from allop- most pronounced in the species with the lowest popula- atry than those in Central Europe. Taken together, this tion density. The collared flycatcher constitutes 60–70 renders reinforcement less likely on the islands than in and 90% of the flycatcher population in the Swedish and the continental hybrid zones (Servedio & Kirkpatrick, the Czech hybrid zones respectively (Eriksson, 1991; 1997; Sætre et al., 1997a). Furthermore, the Swedish Veen et al., 2001). Thus, the density of the pied flycatcher collared flycatcher population is likely to have a Central relative to the collared flycatcher is higher in the Swedish European origin where it is sympatric with the pied than in the Czech population. Consequently, pied fly- flycatcher (Lundberg & Alatalo, 1992). Thus any adapta- catchers in the Swedish hybrid zone should, if anything, tion in the Swedish population of the collared flycatchers be less biased in exposure to heterospecific song, contrary may have its origin in the sympatric Central Europe to our findings. Furthermore, if the frequency of mixed populations. These geographic and demographic patterns singing was only related to population densities we would suggest that adaptations to coexistence would have expect to observe it also in the collared flycatcher, at least evolved further in the Czech than in the Swedish occasionally. However, mixed singing has never been population of the pied flycatcher. Collared flycatchers, reported in the collared flycatcher. Thus, different den- however, could be expected to show similar patterns in sities do not seem to be sufficient to explain this both hybrid zones because of common origin. asymmetry between the species or between the hybrid The variation in male plumage traits is consistent with zones. One explanation could be that pied flycatcher have the above mentioned predictions (Sætre et al., 2003). rudimentary collared flycatcher structures in their song- Collared flycatchers are similarly black and white in the learning predisposition (Eriksson, 1991). The collared two hybrid zones, but Central European pied flycatchers flycatcher might already have diverged to such an extent are more brownish and have smaller patches of white that it is unable to learn the song of the pied flycatcher. compared with pied flycatchers found in the Swedish Song has been suggested to play an important role in hybrid zone (i.e. they differ more from collared flycatch- the development of reproductive barriers in the early ers). As the pied flycatcher is more similar to the collared stage of speciation (Nottebohm, 1972; Grant & Grant, flycatcher in the Swedish hybrid zone, the species 1997a). In oscine passerines song is usually learned differences are larger in the Czech than in the Swedish through an imprinting-like process (Kroodsma, 1982). hybrid zone. If song plays a role in species recognition Such culturally inherited traits may diverge faster than and speciation we could expect to find a similar pattern other genetically determined traits, thus increasing the of variation. rate of allopatric speciation (Grant & Grant, 1997a). It is The song analyses show that the collared flycatcher unclear whether this property of song promotes or does show similar patterns of variation in both hybrid opposes speciation in sympatry (Raikow, 1986; Baptista

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& Trail, 1992; Salomon & Hemim, 1992; Laland, 1994; may evolve. These traits are most likely genetically Grant & Grant, 1997a; Irwin & Price, 1999; Irwin et al., determined, such as plumage traits. However, song may 2001). This plasticity has led researchers to suggest that also have a genetic component affecting its plasticity and song may not respond through reinforcement (Grant & its expression. Accordingly, reinforcement may have Grant, 1997b). Indeed, there is little evidence to support caused innate auditory templates to diverge, such as in the prediction of increased species differences in sympa- the collared flycatcher, and reduce its plasticity, such as try compared with allopatry in bird song (but see the extent of heterospecific copying in the pied flycat- Salomon & Hemim, 1992). Rather, heterospecific copy- cher. Thus, over time, the frequency of maladaptive ing seems to occur when two closely related species come hybridization will decrease, as the species differences in to live in sympatry (Helb et al., 1985). However, even traits used in species recognition increase. Reproductive culturally inherited traits, such as song in passerines, are barriers seem to have evolved further in the Czech than under genetic constraints by an innate auditory song in the Swedish hybrid zones. Thus, we suggest that the template (Kroodsma, 1982). Experiments have shown two different hybrid zones presented in this study may that the song template of birds is affected by both represent different stages in the speciation process. It imprinting and genetics (Marler & Peters, 1977; could be speculated that if heterospecific copying increa- Mundinger, 1995). The effect of imprinting has been ses the frequency of hybridization, it may increase the found to vary even between closely related species selective pressure for reinforcement of prezygotic barri- (Slagsvold et al., 2002). Thus, it seems likely that a ers. Thus, culturally determined traits used in species genetic mechanism determining the plasticity of a trait recognition may, under certain circumstances, function may be under selection (Grant & Grant, 1997c; Irwin & as a catalyst in speciation. Price, 1999). Thus, reinforcement may have caused a divergence of the innate auditory template of sympatric Acknowledgments pied and collared flycatchers. When two species come into secondary contact, species We thank C. Berg, J. Baarman, Centro Studi Ecologici recognition mechanisms may be so similar that hybridi- Appenninici and K. Ra¨sa¨nen for assistance in the field. zation occurs. We suggest that at this early stage of K. Lindroos, P. Na´dvornı´k, A.-C. Syva¨nen and for help in speciation, traits used in species recognition that are the laboratory. For help with statistical analysis and culturally inherited may converge because of hetero- comments on the manuscript we thank S. A. Sæther. specific copying (Fig. 7). Our data suggest that hetero- Permissions to work in the field areas were given by specific copying in pied flycatchers caused the species J. Done´s (Spain) and Centro Studi Ecologici Appenninici differences in song to decrease. If hybrids have greatly (Italy). The study was supported by a grant from The reduced fitness, traits that increase assortative mating Research Council of Norway (JH), The Swedish Research Council (GPS), O. & L. Lamms Memorial Foundation (TB), Uddenberg-Nordingska Foundation (TB & JH), K. & Secondary contact A. Wallenberg Foundation (TB & JH), Wenner-Gren Foundation (GPS), the Spanish Ministry of Science and Technology (JM) and GACR and the Czech Ministry of Education (both SB). We would also like to thank M. Salomon and one anonymous reviewer for comments on the manuscript.

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